CN1529815A - Detection of analytes - Google Patents

Detection of analytes Download PDF

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Publication number
CN1529815A
CN1529815A CNA028060075A CN02806007A CN1529815A CN 1529815 A CN1529815 A CN 1529815A CN A028060075 A CNA028060075 A CN A028060075A CN 02806007 A CN02806007 A CN 02806007A CN 1529815 A CN1529815 A CN 1529815A
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benzyl
analysans
amino
indication mechanism
dimethylamino
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乔治・Y.・丹尼洛夫
乔治·Y.·丹尼洛夫
多德・G.・卡利夫仁特诺斯
亚里士多德·G.·卡利夫仁特诺斯
大・V.・尼古拉特奇克
亚历山大·V.·尼古拉特奇克
・F.・厄尔曼
埃德温·F.·厄尔曼
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Sensors for Medecine and Science Inc
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Priority claimed from US09/754,219 external-priority patent/US20020094586A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

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Abstract

Disclosed are methods for detecting analytes with indicator systems which may undergo a molecular configurational change upon exposure to the analyte. The configurational change affects a detectable quality associated with the indicator system, thereby allowing detection of the presence or concentration of the analyte.

Description

The detection of analysans
The related application contrast
The application is that the part of application number 09/754,219 (application on January 5 calendar year 2001) continues.
The research of subsidizing about federal government or the statement of exploitation are inapplicable
Technical field
The present invention relates to the existence of analysans or the detection of concentration.More precisely, the present invention relates to use indication mechanism to detect analysans, described indication mechanism in being exposed to analysans the time molecular structure can change.The detectable character that the change of structure is relevant with indication mechanism with influence can detect the existence or the concentration of analysans thus.
Background technology
United States Patent (USP) 5 503 770 people such as () James relates to a kind of fluorescent chemicals that comprises boric acid, will send high-intensity fluorescence when it is bonded to when carbohydrate (comprising glucose) is gone up.Comprise a fluorophor in the molecular structure of described fluorescent chemicals, at least one phenylboric acid part and at least one supply amine nitrogen atom; Wherein nitrogen-atoms is arranged near the phenylboric acid part, so as with boric acid generation intramolecular interaction.Thus, above-mentioned interaction will make compound emitting fluorescence when combining with carbohydrate.The compound that is suitable for detecting carbohydrate has been described in the United States Patent (USP) 5 503 770.J.Am.Chem.Soc.117 (35) referring to people such as T.James: 8982-87 (1995).
NatureBiotechnology16,49-53 (1998) relate to and utilize molecular beacon, promptly are marked with the right hair clip shape oligonucleotide probes of fluorophor/quencher, carry out allele and differentiate.In the time of on being bonded to object, probe will experience structure reorganization, thereby recover the fluorescence of the fluorophor of inner quencher.Yet, because the intensity of DNA base pairing is higher relatively at ambient temperature, therefore, when using, molecular beacon probe must experience big structural change (by 180 ° change basically), and described system can not easily be used for detecting in real time continuously the fluctuation of analyte concentration.
Therefore, also has such demand for indication mechanism in the prior art, be that described system can detect existing or concentration of analysans with higher sensitivity, it can also use multiple detection system, and can be used for the analysans that concentration can fluctuate is detected in real time.
Summary of the invention
On the one hand, the present invention relates to the existence of polyhydroxy analysans in a kind of test samples or the method for concentration, this method comprises:
A) sample is exposed in the indication mechanism, described system has:
I) can be with first recognition unit of reversible manner and described analysans formation covalent bond, with can form second recognition unit (A) of covalent bond with reversible manner and the described analysans that is bonded on first recognition unit, or can be when not having described analysans with reversible manner and the interactional ligand of first recognition unit unit (B); Described ligand unit comprises the mark that produces detectability also not essentially, described detectability is regulated and control by the interaction of ligand unit and recognition unit, and the part that wherein comprises described first recognition unit in the indication mechanism is connected on the indication mechanism part that comprises described second recognition unit or described ligand unit with covalent manner or non-covalent mode; With
Ii) comprise following detection system one of at least: (A) produce the donor/acceptor system that can detect characteristic, when described indication mechanism is exposed in the described analysans, this system changes in the mode of concentration-dependence, or (B) the described ligand unit that is labeled; With
B) measure any change of described detection characteristic, be determined at the existence or the concentration of analysans described in the described sample thus.
On the other hand, the present invention relates to implement the indication mechanism of said method.
Brief description
Fig. 1 shows the standardization fluorescent emission (at the I/Io of 535nm) of the compound of describing among the embodiment 1.
Fig. 2 shows the standardization fluorescent emission (at the I/Io of 535nm) of the compound of describing among the embodiment 2.
Fig. 3 shows the fluorescent emission (at the I of 535nm) of the indication mechanism of describing among the embodiment 3.
Fig. 4 shows the fluorescent emission (at the I of 535nm) of the indication mechanism of describing among the embodiment 4.
Fig. 5 shows the fluorescent emission (at the I of 535nm) of the indication mechanism of describing among the embodiment 5.
Fig. 6 shows the fluorescent emission (at the I of 450nm) of the indication mechanism of describing among the embodiment 6.
Fig. 7 shows the standardization fluorescent emission (at the I of 430nm) of the indication mechanism of describing among the embodiment 6.
Fig. 8 shows the absorption spectrum of the indication mechanism of describing among the embodiment 7.
Fig. 9 shows the ratio (A (565nm)/A (430nm)) of the indication mechanism absorptance of describing among the embodiment 7.
Figure 10 shows the standardization fluorescence (I/Io) of the indication mechanism of description among the embodiment 7 at 550nm.
Detailed Description Of The Invention
On the one hand, utilize the indication system that the meeting recurring structure changes when interacting with analysans System the invention provides a kind of existence of analysans or method of concentration of detecting. When described finger When showing system's experience structural change, its detected characteristic can change, and this can indicate and treat The existence of analyte or change in concentration.
Can detect many analysans according to the present invention. Suitable analysans comprises The molecule analysans (opposite with the metal ion or the metal complex that are for example formed by coordinate bond, it Can be called the molecule that is formed by covalent bond); Carbohydrate; Polyol, especially Those compounds with vicinal hydroxyl are such as free sugar (for example glucose, fructose, lactose etc.) With the sugar that is bonded on lipoid, the protein etc.; Small-molecule drug; Hormone; Oxygen; Carbon dioxide; Various ions are such as zinc ion, potassium ion, hydrogen ion, carbanion etc. The present invention Be particularly suited for detecting the small-molecular weight analysans, particularly daltonian to be analyzed less than 5000 Thing.
In one embodiment, the present invention can utilize for detected analyte and have at least The indication mechanism of two recognition units is implemented, and it can and be treated on two sites by orientation Analyte interacts, thereby the indication mechanism recurring structure is changed. In addition, described finger Show that system also has the detection system that is mated, when indication mechanism and analysans interaction The time, the detected characteristic that detection system has changes. When interacting with analysans, Recognition unit can present a configuration, and the configuration when not having analysans is compared, at new structure They will draw closer together or a good distance away in the type, perhaps limit the free degree of its molecular motion, this Can exert an influence to signal again. The change of configuration can cause the change that can detect characteristic.
In another embodiment, implement the used indication mechanism of the present invention and have at least one pin Recognition unit and a ligand unit to analyte to be detected. Ligand unit and identification form Reversible interaction can take place between the unit, and with analysans and recognition unit mutually Effect is competition mutually. Do not having when recognition unit and ligand unit in the situation of analysans mutually Do the time spent, detection system will have different preferred configuration or relative orientation, and at analysans When interacting with recognition unit, the ligand unit will be replaced from recognition unit. The change of configuration can cause the change that can detect characteristic. In certain embodiments, ligand Unit further is the part of detection system. For example, the ligand unit also can be quencher, Its effect will be eliminated when analysans and recognition unit interaction. In addition, ligand is single Unit for example can also comprise: characteristic (for example spatial distribution) with ligand unit and recognition unit it Between interactional existence whether and different detectable labels.
For above-described arbitrary embodiment, suitable recognition unit comprise preferably can with The reversibly interactional Molecular fragments of detected analyte. Be understood that term " the mutual work With " can comprise physics miscellaneous and interaction chemistry, as charge interaction, Hydrogen bonding, covalent bonding etc. Particularly preferably be, between recognition unit and analysans Interaction, and the phase between ligand unit (if present) and recognition unit Mutual effect, form for to form one or more covalent bond with reversible manner. At this, altogether Valence link preferably refers to two keys between the atom, and wherein each atom provides an electronics, And do not comprise hydrogen bonding, ionic bonding and the coordinate bond of two electronics is provided by one of two atoms Close or the coordination valence bonding. Preferably more weak interaction, for example dissociation constant is higher than about 10-6M. Known suitable recognition unit has multiple, preferably include boric acid, borate ion, arsenious acid, Arsenous anion ion, telluric acid, tellurate radical ion, germanic acid, germanic acid radical ion etc., known institute State these and can be used for identifying vicinal diols such as glucose and other carbohydrate. Work as analysans When being glucose, boric acid is most preferred recognition unit.
Comprise that in indication mechanism in the embodiment of ligand unit, such unit should Interact with recognition unit, and design according to the dynamic range of target analysans. Under above-mentioned guideline, the selection of ligand unit will be depended on analysans and identification form Unit. In preferred embodiments, when analysans is vicinal diols such as glucose, and know When other unit was boric acid, the ligand unit preferably can be with reversible manner and recognition unit Cheng Jian Molecular fragments (such as ester bond). Above-mentioned ligand unit comprise aromatic diol (for example catechol), Lactate, alpha-hydroxy acid, tartaric acid, malic acid, diethanol amine, beta-alkamine, glucose, Polyol and the compound that contains vicinal hydroxyl groups, described these materials can be replace or Unsubstituted. In another embodiment, the ligand unit further is one of detection system Part. For example, the glimmering of the fluorophor that associates with indication mechanism can also be regulated and control in the ligand unit Light. When ligand unit and recognition unit interaction, it will be in for example can be effectively The configuration of quench fluorescence group. When the ligand unit is replaced lower by analysans from recognition unit When coming, ligand no longer is the configuration (referring to embodiment 6) of quench fluorescence group. Real at another Execute in the scheme, opposite situation (quencher when interacting with recognition unit can also be arranged really Can not interact with fluorophor).
In the use, indication mechanism of the present invention preferably is among the mobile equilibrium of said configuration state.More preferably, there are more weak combination and interaction at a high speed, make and in the presence of free analysans, can realize balance faster.Therefore, use the present invention in very wide condition and range, to detect in real time, especially detect the fluctuation of concentration of analysans analysans.Usually, when implementing method described herein, the present invention does not need to use big temperature change.That is to say that method of the present invention can be carried out basically at ambient temperature, this means the temperature of finding the analysans sample under the normal condition.Very clear, environment temperature will change widely according to analysans and environment thereof.For example, environment temperature can comprise room temperature or colder; Can be for many intravital application up to about 45 ℃; Can be and use up to about 80 ℃ or higher for for example some fermentation.
Indication mechanism of the present invention comprises detection system, and when indication mechanism was exposed in the analysans, this detection system had the detected characteristic that the mode with concentration-dependence changes.Detection system preferably comprises to body/receptor system, and it refers to a pair of different group that signal is provided by interacting, and wherein the change of distance will change the characteristic of signal between the group.Preferably, described signal be electromagnetism or electrochemical signals (for example, when closely near the time provide the electric charge of different electrochemical potentials shift to).
There are many kinds in known above-mentioned character/system, and can be used for the present invention.For example, indication mechanism can comprise luminous (fluorescence or phosphorescent) or chemiluminescent mark, and based on mark of absorptance or the like, when the structural change of indication mechanism experience, the detection characteristic of described mark will change.Detection system can comprise to body portion and acceptor portion, and is at regular intervals separately, so that detectable change is arranged when indication mechanism and analysans interaction.
Detecting characteristic can be that detectable spectrum changes, as the change (determining by time domain or frequency domain measurement) of fluorescence decay time, the change of fluorescence intensity, the change of fluorescence anisotropy or polarization; The spectrum of emission spectrum moves; Time-change (determining) of anisotropy decay of resolution by time domain or frequency domain measurement, the change of absorption spectrum, or the like.
Detection system can comprise the molecule segment of fluorophor and fluorescence that can the quench fluorescence group.In this embodiment, indication mechanism can constitute in two ways.At first, it can constitute like this, and to cause when not having analysans, fluorophor and quencher mutual distance are very near, the result effectively quencher the emission of fluorescence.When interacting with analysans, the configuration of indication mechanism changes, and causing is enough to make fluorophor to go the separation of the fluorophor/quencher of quencher (dequenching).In addition, indication mechanism can constitute like this, and to cause when not having analysans, fluorophor and quencher mutual distance are far, and this moment, fluorophor can emitting fluorescence.When interacting with analysans, the configuration of indication mechanism changes, and makes fluorophor/quencher enough approaching, thereby with the fluorophor quencher.Fluorophor/the quencher of Shi Yonging is to comprising following situation in the present invention: the right both sides of described fluorophor/quencher are identical or different fluorophor, but when indication mechanism is in the quencher configuration, as shifting or the like by closing effect, energy, a fluorophor will influence another fluorescence.
Known fluorophor/quencher is many to having, and it is right to be that the present invention has also designed a plurality of fluorophor/quenchers.For example, be known that the DABCYL many fluorophor of quencher effectively, as cumarin, EDANS, fluorescein, lucifer yellow, BODIPY TMEosine, tetramethyl rhodamine, Texas Red TMOr the like.
It should be understood that from the fluorophor emitted fluorescence and can come quencher by multiple mechanism.A kind of is the quencher by photic electron transfer between fluorophor and the quencher (referring to Acc.Chem.Res.1994,27,302-308 is introduced into as a reference at this).Quencher can also be by by the caused intersystem transition of heavy atom effect or by carrying out with the interaction of paramagnetic metal ion, and wherein quencher can comprise heavy atom such as iodine or paramagnetic metal ion such as Cu + 2(referring to for example J.Am.Chem.Soc.1985,107,7783-7784 and J.Chem.Soc.Faraday Trans., 1992,88,2129-2137, both all are incorporated herein by reference).
Quencher can also take place by form the ground state complex between fluorophor and quencher, as Nature Biotechnology, and 1998,16, described in the 49-53 (being hereby incorporated by).Another quenching mechanism for example relates at Meas.Sci.Technol.10 (1999) 127-136 and JACS2000, and 122, the fluorescence resonance energy transmission of describing among the 10466-10467 (being hereby incorporated by) (FRET).
The another kind of molecule segment useful to detection system of the present invention comprises: absorption spectrum changes those molecule segments that change with molecular configuration, comprises Alizarin Red-S, or the like.
The indication mechanism that suitable the present invention uses comprises the composition of the material with one of following array structure:
Figure A0280600700131
Or
R 1-D 1-L 1-Z-L 2-D 2-R 2
Or
D 1-R 1-L 1-Z-L 2-R 2-D 2
In the formula:
-R 1Be one or more recognition units at described analysans;
-R 2Be i) at one or more recognition units of described analysans, or the ii) dispensable ligand unit that is labeled;
-D 1And D 2Comprise detection system together, detection system comprises energy and gives body/receptor system, and when described indicator molecules and analysans interaction, this detection system has the detection characteristic that changes in concentration-dependence mode; Perhaps work as R 2When being the ligand unit that is labeled, D 1And D 2Can not exist;
-L 1And L 2Identical or different, and the length of the linking group that it comprised and structure are enough to make the variation that interacts and can detect characteristic to take place; With
Z is at L 1And L 2Between covalent bond or non--covalent bond.
Recognition unit, ligand unit and detection system had been described already.Linking group L 1And L 2Having is enough to make described interaction and changes the length and the structure that can take place.Be recognized that the exact nature of linking group will depend on the structure of other unit of indication mechanism.Can design coupling agent with regard to the rigidity of structure, molecule distance, charge interaction etc., as shown in the Examples, this can be used to make the interaction optimization of reversible analysans detection system.The Z component of indication mechanism of the present invention is preferably at L 1And L 2Between covalent bond.Indication mechanism can be unimolecule or macromolecular form.
L 1And L 2Can take multiple form.For example, suitable linking group comprises alkyl, aryl, polyamide, polyethers, poly-amino, polyester and combination thereof, and described these groups are replacements or unsubstituted.
Indication mechanism of the present invention is if soluble words can directly be used in solution as required.On the other hand, if necessary, indication mechanism can be fixed (as by mechanical entrapment or connection covalency or ion) to as on insoluble surface such as glass, plastics, polymeric material or the matrix or its inside.When indication mechanism for example is entrained in polymkeric substance when inner, support materials preferably can fully be permeated for analysans, so that suitable interaction is provided between analysans and indication mechanism.
If indication mechanism is slightly soluble in water or water insoluble, still wish the words that in aqueous medium, detect, can make indication mechanism and hydrophilic monomer copolymerization, thereby form as await the reply jointly U. S. application 09/632, the described hydrophilic big molecule of 624 (applications on August 4th, 2000) is introduced into as a reference at this.
Be understandable that indication mechanism of the present invention chemically can take many forms.For example, whole indication mechanism can be a molecule that size is less.Perhaps, the individual components of indication mechanism can be a macromolecular part.Under one situation of back, each component of system can be mixed in the identical polymkeric substance, perhaps can with independent crosslinked polymer associate.For example, comprising the independent monomer of fluorophor/ligand unit adduct and quencher/recognition unit adduct can copolymerization and form indication mechanism polymkeric substance (referring to embodiment 5).In addition, can make monomer polymerization form independent polymer chain individually, and then crosslinked and form indication mechanism.
Indication mechanism of the present invention has many application, is included in the energy, medicine and agriculture aspect as indicator.For example, indication mechanism can be used for detecting the detection of blood or urine trace or ultramicron glucose as indicator molecules, and therefore, the disease as diabetes and adrenal insufficiency provides valuable information for diagnosis or monitoring.Indication mechanism of the present invention with two recognition units especially can be used for detecting the glucose in the solution, and the alpha-hydroxy acid or the beta-diketon that wherein can also comprise interference volume potentially (are applied for 09/754,217 (application on January 5 calendar year 2001) referring to awaiting the reply jointly; 60/329,746 (application on October 18 calendar year 2001); With 60/269,887 (application on February 21 calendar year 2001), denomination of invention is " to the detection of glucose in the solution that contains alpha-hydroxy acid or beta-diketon ", is introduced into as a reference at this).The production that is used for the medical/medicinal glucose of health care aspect needs monitoring.
The present invention's application agriculturally comprises detection analysans such as glucose, the content in soybean and other agricultural product.For the high-value product as European grape, before making the results decision-making, must carefully monitor glucose.Because glucose is carbon source and raw material the most expensive in the sweat, therefore, in strong drink is produced, in order to make reactor feed rate controlled optimization, be crucial to the monitoring of glucose.At soft drink and alcoholic beverage production period, the mixing of reactant and the control of concentration of glucose also are vital for quality control, and from the whole world, the glucose and the fermentable sugars (cis-glycol) of the overwhelming majority are used to above-mentioned production.
When being attached to the fluorescence indicator substituting group in the detection system, system of the present invention can be used for known various detection techniques.For example, system of the present invention can be used for fluorescence sensing device (for example United States Patent (USP) 5 517 313) maybe can be bonded to polymeric material, as is used for macroscopic test paper.Back one technology for example makes the measurement of glucose be similar to litmus paper bar measurement pH.System described herein can also use as fluorescence spectrometer or clinical analysers by manufacturings such as Shimadzu, Hitachi, Jasco, Beckman as simple reagent small-sized (benchtop) analysis and test device with standard.Also that analysans is the specific chemistry/optical signalling of these molecules by conversion offer as by Ocean Optics (Dunedin, Florida) or OrielOptics make based on fibre-optic sensor and analysis of fluorescence meter.
The United States Patent (USP) 5 517 313 that is hereby incorporated by has been described: can use the fluorescence sense equipment of system of the present invention, it can be used to measure the glucose in analysans such as the fluid nutrient medium or the existence or the concentration of other cis-diol compounds.This sensing equipment comprises: the matrix (below be referred to as " fluorescence matrix "), high flux filtrator and the photodetector that comprise the fluorescence indication mechanism that stratification arranges.In this device, light source, preferred light emitting diode (" LED ") is positioned at indicator material, perhaps in the waveguide below the indicator matrix, so that from the incident light of light source indication mechanism is fluoresced at least in part.The high flux filtrator makes emission luminous energy get at and reach photodetector, can filter out the incident light of dispersion simultaneously from light source.
Use the analysans of inhomogeneous existence, as glucose or other cis-diol compounds, the fluorescence of the indicator molecules that uses in the device to description in the United States Patent (USP) 5 517 313 is regulated and control, and for example weakens or strengthens.
In the sensor of describing in United States Patent (USP) 5 517 313, the material that comprises indicator can permeate analysans.Therefore, analysans can diffuse into this material from test(ing) medium on every side, influences thus by the indication mechanism emitted fluorescence.By setting to light source, the material that comprises indication mechanism, high flux filtrator and photodetector, make at least a portion fluorescence directive photodetector of indication mechanism emission, thereby generation electric signal, this signal are exactly the indication of analysans in the surrounding medium (for example glucose) concentration.
According to other possible embodiments of utilizing indication mechanism of the present invention, sensing equipment also is described in United States Patent (USP) 5 910 661,5 917 605 and 5 894 351, is introduced into as a reference at this.
System of the present invention also can be used in the implanted device, for example monitors the analysans (as the blood sugar amount) in the body continuously.Suitable unit describe is in for example, the patented claim United States Patent (USP) 09/,383 148 that awaits the reply jointly (application on August 26th, 1999), and in United States Patent (USP) 5 833 603,6 002954 and 6 011 984, be introduced into as a reference at this.
Utilize known reaction mechanism and reagent, comprise the reaction mechanism consistent with general procedure as described below, need not excessive test, those skilled in the art can prepare system of the present invention.
Embodiment 1
NBuF-dimethylbenzene-Q two-borate nBuF-dimethylbenzene-Q two-borate nBuF-dimethylbenzene-Q two-borate N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono) benzyl]-amino hexyl]-[2-(borono) benzyl] aminoethyl-4-butyl amino-1,8-naphthalimide (nBuF-hexa-Qbis-boronate).
The free hypoboric acid product that uses in glucose research is by with N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(5,5-dimethyl borine-2-yl) benzyl] amino hexyl]-[2-(5,5-dimethyl borine-2-yl) benzyl] aminoethyl-4-butyl amino-1, the 8-naphthalimide is dissolved in the MeOH/PBS buffer system and obtains.
N-(2,2-diethoxy ethyl)-4-bromo-1, the 8-naphthalimide
At 45 ℃, with 4-bromo-1,8-naphthalic anhydride (10.0 grams, 36.1 mMs) and the suspending liquid of aminoacetaldehyde diethyl acetal (4.81 grams, 5.26 milliliters, 36.1 mMs, 1 gram equivalent) in 45 milliliters of EtOH stirred 3 days.Filter the suspending liquid that obtains then, wash with EtOH, and residue is carried out drying, obtain 13.3 gram (94%) light brown solid products.
TLC:Merck silica gel 60 plates, Rf 0.17 (98/2 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mm NovaPak HR C18 post injects 0.050 milliliter, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 360nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 24.2 minutes residence time.
Figure A0280600700182
N-(2,2-diethoxy ethyl)-4-fourth amino-1, the 8-naphthalimide
Under 45 ℃, with N-(2,2-diethoxy ethyl)-4-bromo-1,8-naphthalimide (0.797 gram, 2.03 mMs) and the solution heating of normal-butyl ammonia (1.48 grams, 2.00 milliliters, 20.2 mMs, 9.96 gram equivalents) in 8 milliliters of NMP 66 hours.At this moment, the suspending liquid that obtains is cooled to 25 ℃, succeeded by filtration.With 50 milliliters of ether dissolution residues and utilize 3 * 50 milliliters water to extract.On anhydrous sodium sulfate, organic extract is carried out drying, filter and concentrate, so that obtain thick yellow powder.By silica gel chromatography (25 grammes per square metre power level gels, 0-1%CH 3OH/CH 2Cl 2) this raw material is purified, thereby obtain 0.639 the gram (82%) yellow powder.
TLC:Merck silica gel 60 plates, Rf 0.71 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mm NovaPak HR C18 post injects 0.050 milliliter, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 23.5 minutes residence time.
N-(2-oxygen ethyl)-4-butyl bromide-1, the 8-naphthalimide
At 25 ℃, the solution stirring in 25 milliliters of acetone is 18 hours with N-(2,2-diethoxy ethyl)-4-butyl bromide-1,8 naphthalimide (0.622 gram, 1.62 mMs) and right-toluenesulfonic acid monohydrate (0.010 gram, 0.053 mM, 0.032 gram equivalent).At this moment, this solution is concentrated and by silica gel chromatography (25 grammes per square metre power level gels, 0-1%CH 3OH/CH 2Cl 2) residue is purified, thereby obtain 0.47 the gram (94%) orange solids.
TLC:Merck silica gel 60 plates, Rf 0.61 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
1H?NMR(400MHz，CDCl 3)；δ1.03(t，3H，J＝7.3Hz)，1.53(m，2H)，1.78(m，2H)，3.38(t，2H，J＝7.2Hz)，5.02(s，2H)，6.64(d，1H，J＝8.6Hz)，7.52(dd，1H，J＝7.4，8.3Hz)，8.08(dd，1H，J＝1Hz，8.5Hz)，8.38(d，1H，J＝8.3Hz)，8.46(dd，1H，J＝1.0，7.3Hz)，9.75(s，1H).
HPLC:HP 1100HPLC chromatograph, water 5 * 100mm NovaPak HR C18 post injects 0.050 milliliter, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 19.6 minutes residence time.
N-(4-dimethylamino benzyl)-1, the 6-diamino hexane
Under 25 ℃, in the dark, under nitrogen atmosphere, with 4-dimethylaminobenzaldehyde (1.00 grams, 6.70 mMs), sodium sulphate (6.7 grams, 47.2 mM, 7.04 gram equivalents) and 1,6-diamino hexane (3.89 grams, 33.5 mM, 5.00 gram equivalents) suspending liquid in 20 milliliters of anhydrous EtOH stirred 18 hours.At this moment, solution is filtered and with NaBH 4(1.73 grams, 45.8 mMs, 6.84 gram equivalents) are added in the filtrate.Under 25 ℃, suspending liquid was stirred 5 hours.At this moment, reaction mixture is concentrated, and residue is dissolved in 50 ml waters, use 3 * 50 milliliters of ether to extract again.With 2 * 50 ml waters the organic extract that mixes is washed.With 2 * 50 milliliters of ether the aqueous extract that mixes is extracted.On anhydrous sodium sulfate, the organic extract that mixes is carried out drying, filter and concentrate, so that obtain viscous oil (1.35 gram (81%)).
TLC:Merck silica gel 60 plates, Rf0.58 (80/15/5 CH 2Cl 2/ CH 3OH/iPrNH 2), utilize triketohydrindene hydrate colorant, UV (254/366) to observe.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mm NovaPak HR C18 post, 0.050 milliliter of injection, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 280nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 13.3 minutes residence time.
Figure A0280600700211
N-2-[5-(N-4-dimethylamino benzyl) ammonia hexyl] aminoethyl)-4-fourth amino-1, the 8-naphthalimide.
To N-(2-oxygen ethyl)-4-fourth amino-1,8-naphthalimide (0.346 gram, 1.11 mM) add N-(4-dimethylamino benzyl) 1 in the suspending liquid in 25 milliliters of absolute methanols, 6-diamino hexane (0.554 gram, 2.22 mM, 2.00 gram equivalent) and the solution of acetate (0.067 gram, 1.11 mMs, 1.0 gram equivalents) in 20 milliliters of absolute methanols.In this potpourri, add NaCNBH 3(0.070 gram, 1,1 mM, 1.0 gram equivalents) solution in 5 milliliters of absolute methanols.At 25 ℃ reaction mixture was stirred 15 hours.At this moment, remove methyl alcohol, and residue is dissolved in 30 ml waters by rotary evaporation.Utilize the pH regulator to 2 of the HCl of 1N, stirred 1 hour at 25 ℃ then solution.At this moment, utilize the pH regulator to 12 of the NaOH of 1N, use 3 * 50 milliliters of CH then solution 2Cl 2Extract.With 3 * 50 ml waters the organic extract that mixes is washed, on anhydrous sodium sulfate, carry out drying, filter and concentrate, so that obtain rough brown oil.By silica gel chromatography (35 gram flash distillation level gels, 0-50%CH 3OH/CH 2Cl 2, 45/50/5CH then 3OH/CH 2Cl 2/ iPrNH 2) this raw material is purified, thereby obtain 0.190 gram (32%) diamines product.
FAB MS: for C 33H 45N 5O 2, calculated value [M] +554; Measured value [M] +554.
TLC:Merck silica gel 60 plates, Rf 0.42 (80/20CH 2Cl 2/ CH 3OH), utilize triketohydrindene hydrate colorant and UV (254/366) to observe.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mm NovaPak HR C18 post, 0.050 milliliter of injection, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 17.6 minutes residence time.
N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(5,5-dimethyl borine-2-yl) benzyl] amino hexyl]-[2-(5,5-dimethyl borine-2-yl) benzyl] aminoethyl-4-fourth amino-1, the 8-naphthalimide.
To N-2-[5-(N-4-dimethylamino-benzyl) ammonia hexane] aminoethyl)-4-fourth amino-1,8-naphthalimide (0.150 gram, 0.276 mM) and DIEA (0.355 the gram, 0.478 milliliter, 2.81 mM, 10.0 gram equivalent) add (2 bromoethyl benzene base) boric acid neopentyl ester (0.390 gram, 1.38 mMs, 5.00 gram equivalents) in the solution in 5 milliliters of methenyl cholorides at 2 milliliters of CHCl 3In solution.Then, at 25 ℃ to this solution stirring 27 hours.At this moment, this potpourri is concentrated and by alumina column chromatography (neutral alumina that 100 grams activate, 0-5%CH 3OH/CH 2Cl 2) residue is purified, thereby obtain 0.024 the gram (19%) the viscosity brown oil.
FAB MS (glycerine matrix): for C 53H 67B 2N 5O 8, calculated value is [M] +924 (two glycerine adducts substitute the di neo-pentyl ester of boric acid); Measured value is [M] +924
TLC:Merck neutral alumina plate, Rf0.62 (80/20CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mm NovaPak HR C18 post, 0.050 milliliter of injection, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 20.7 minutes residence time.
NBuF-dimethylbenzene-Q two-borate:
N-2-[4-(N-4-dimethylamino benzyl)-[2-(borono) benzyl] amino-methyl] benzyl-[2-(borono) benzyl] aminoethyl-4-fourth amino-1,8-naphthalimide (nBuF-dimethylbenzene-Q two-borate)
With 1-[N-(4-dimethylamino benzyl) amino] methyl-4-aminomethyl phenyl is as diamines coupling spouse, employing is similar to N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono) benzyl]-the ammonia hexyl]-[2-(borono) benzyl] aminoethyl-4-fourth amino-1, the mode of 8-naphthalimide (nBuF-hexa-Q-hypoboric acid ester) prepares described compound.
The contrast indicator molecules:
NBuF list-borate:
N-2-(ethyloic)-2-[2-(borono) benzyl] aminoethyl-4-fourth amino-1,8-naphthalimide (nBuF list-borate)
Figure A0280600700242
N-2-(tert-butoxycarbonyl) aminoethyl-4-bromo-1, the 8-naphthalimide
At 45 ℃, with 4-bromo-1,8-naphthalic anhydride (1.00 grams, 3.61 mMs) and N-(uncle-butoxy carbonyl)-1, the suspending liquid of 2-diaminoethanes (0.578 gram, 3.61 mMs, 1.00 gram equivalents) in 20 milliliters of EtOH stirred 2 hours.At this moment, in 15 minutes, temperature is risen to 150 ℃.Subsequently, making reaction mixture be cooled to 25 ℃ also stirred 15 hours again.At this moment wait, with the suspension filtered that obtains, wash, and residue is carried out drying, obtain 1.03 gram (68%) light brown solid products with EtOH.
TLC:Merck silica gel 60 plates, Rf0.63 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
Figure A0280600700251
N-2-(uncle-butoxy carbonyl) aminoethyl-4-fourth amino-1, the 8-naphthalimide
Under 45 ℃, with N-(uncle-butoxy carbonyl) aminoethyl-4-bromo-1,8-naphthalimide (0.900 gram, 2.15 mMs) and the just-solution of butyl ammonia (0.786 gram, 1.06 milliliters, 10.7 mMs, 5.01 gram equivalents) in 5 milliliters of NMP heated 17 hours.At this moment wait, adds second part just-butylamine (0.786 restrains, 1.06 milliliters, 10.7 mMs, 5.01 gram equivalents).At 25 ℃ to the solution stirring that obtains 23 hours.At this moment, under vacuum, potpourri is concentrated.By silica gel chromatography (50 grammes per square metre power level gels, 0%, 4%CH then 3OH/CH 2Cl 2The segmentation gradient) residue is purified, thereby obtain the viscosity yellow solid that 0.97 gram contains remaining NMP.According to present appearance, this material is proceeded test.
FAB MS: for C 23H 29N 3O 4, calculated value is [M]+411; Measured value is [M]+411.
TLC:Merck silica gel 60 plates, Rf 0.60 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
N-2-aminoethyl-4-fourth amino-1,8-naphthalimide list trifluoroacetate
At 25 ℃, with N-2-(uncle-butoxy carbonyl) aminoethyl-4-bromo-1,8-naphthalimide (0.92 gram, 2.24 mMs) is at 20 milliliters of 20% trifluoroacetic acid/CH 2Cl 2In solution stirring 19 hours.At this moment, flow down at nitrogen reaction mixture is concentrated.Utilize ether that residue is pulverized, and the solid that obtains is dry in a vacuum, to obtain 0.772 gram (81%) orange powder.
FAB MS: for C 18H 21N 3O 2, calculated value is [M] +311; Measured value is [M] +312.
HPLC:HP 1100HPLC chromatograph, Vydac 201TP 10 * 250mm post, 0.100 milliliter of injection, 2 ml/min, detect wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B 2 minutes, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 19.5 minutes residence time.
Figure A0280600700261
Uncle N-2-[(-butoxy carbonyl) methyl] aminoethyl-4-fourth amino-1, the 8-naphthalimide
At 25 ℃, with N-2-aminoethyl-4-fourth amino-1,8-naphthalimide list trifluoroacetate (0.99 gram, 0.23 mM), DIEA (0.167 gram, 0.225 milliliter, 1.29 mM, 5.55 gram equivalents) and bromoacetic acid uncle-butyl ester (0.032 gram, 0.024 milliliter, 0.16 mM, 0.70 gram equivalent) at 2.5 milliliters of CH 2Cl 2In solution stirring 23 hours.At this moment wait, add 25 milliliters of CH 2Cl 2, with 1 * 25 milliliter of saturated NaHCO 3This solution is washed, in anhydrous Na 2SO 4Go up organic extract is carried out drying, filter and concentrate.By silica gel chromatography (15 grammes per square metre power level gels, 0%-4%CH 3OH/CH 2Cl 2) residue is purified, thereby obtain 0.051 gram (73%) yellow glass shape solid.
TLC:Merck silica gel 60 plates, Rf 0.27 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe
Uncle N-2-[(-butoxy carbonyl) methyl]-2-[2-(5,5-dimethyl borine-2-yl) benzyl] aminoethyl-4-fourth amino-1, the 8-naphthalimide
At 25 ℃, with uncle N-2-[(-butoxy carbonyl) methyl]-aminoethyl-4-fourth amino-1,8 naphthalimides (0.051 gram, 0.12 mM), DIEA (0.78 gram, 0.11 milliliter, 0.60 mM, 5.0 gram equivalent) and (2 bromo toluene base) boric acid neopentyl ester (0.083 restrain, 0.29 mM, 2.4 gram equivalents) at 10 milliliters of CH 2Cl 2In solution stirring 72 hours.At this moment, this potpourri is concentrated and by silica gel chromatography (10 grammes per square metre power level gels, 0-1%CH 3OH/CH 2Cl 2) purify, thereby obtain 0.035 gram (47%) glass orange solids.According to present appearance, this product is proceeded test.
TLC:Merck silica gel 60 plates, Rf 0.39 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
N-2-(ethyloic)-2-[2-(borono) benzyl] aminoethyl-4-fourth amino-1,8-naphthalimide (nBuF list-borate)
At 25 ℃, with uncle N-2-[(-butoxy carbonyl)-methyl]-2-[2-(5,5-dimethyl borine-2-yl) benzyl] aminoethyl-4-fourth amino-1,8-naphthalimide (0.035 gram, 0.056 mM) is at 5 milliliters of 20%TFA/CH 2Cl 2In solution stirring 16 hours.At this moment wait, flow down this solution concentration at nitrogen, and residue is pulverized, so that obtain orange solids with ether.By silica gel chromatography (8 grammes per square metre power level gels, 0-5%CH 3OH/CH 2Cl 2) this raw material is purified, thereby obtain 0.011 the gram (39%) the yellow/orange solid.
FAB MS: for C 30H 34BN 3O 7, calculated value is [M] +559 (single glycerine adducts); Measured value is [M] +560.
TLC:Merck silica gel 60 plates, Rf 0.26 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
The regulation and control of fluorescence
The regulation and control of glucose have been determined to the fluorescence of three kinds of compounds preparing in the present embodiment.
Fig. 1 shows nBuF-hexa-Q hypoboric acid ester (" hexa-Q ") indicator (0.015mM), nBuF-dimethylbenzene-Q two-borate (" dimethylbenzene Q ") indicator (0.049mM) and the standardized fluorescent emission (I/Io at 535nm) of nBuF list-borate contrast indicator (0.029mM) in comprising the 70/30MeOH/PBS solution of 0-20mM glucose.Utilize Shimadzu RF-5301 spectrofluorimeter spectra re-recorded, wherein be activated at 450nm; Excitation slit 1.5nm; Emission slit 1.5nm; Environment temperature.Error line is the standard deviation of the triple values of each data point.
Data show: the fluorescence of nBuF list-borate indicator compound is not subjected to the influence of the existence of glucose.The fluorescence of NBuF-dimethylbenzene-Q hypoboric acid ester indicator compound is subjected to the influence of glucose more or less, and the fluorescence of nBuF-hexa-Q two-borate indicator compound is subjected to the influence of glucose widely in the scope of 0-5mM.It is believed that is not having under the situation of glucose, and pliable and tough relatively hexa-methylene connects and makes that N-4-dimethylamino benzyl quencher group can be fully near the naphthalimide fluorophor in the hexa-Q compound, thus the quencher latter's fluorescence effectively.Having under the situation of glucose, two boric acid recognition units will participate in glucose and connect, and therefore will change the molecular configuration of indicator, and fluorophor is separated fully with the quencher group, so that make fluorescent emission go quencher (dequenched).Can see same effect for dimethylbenzene-Q compound, but because the dimethylbenzene coupling agent is not too pliable and tough, so its effect degree is much smaller, therefore, when glucose connects, only allows less separation between fluorophor and the quencher group.
But control compounds comprises fluorophor does not have the quencher group.Control compounds is not having emitting fluorescence under the situation of glucose, and its fluorescence is unaffected when adding glucose.
Embodiment 2
Amino ethoxy F-hexa-C two-borate amino ethoxy F-hexa-C two-borate
Figure A0280600700292
N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono) benzyl]-amino hexyl]-[2-(borono) benzyl] aminoethyl-4-[2-(2-amino ethoxy) ethoxyethyl group] amino-1,8-naphthalimide (amino ethoxy F-hexa-Q two-borate)
By following improvement, with being similar to N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono) benzyl]-amino hexyl]-[2-(borono) benzyl] aminoethyl-4-butyl amino-1, the mode of 8-naphthalimide (nBuF-hexa-Qbis-boronate) prepares this compound.After the dibenzyl boronation effect of diamines intermediate was finished, just with 1, the 4-bromine position of 8-naphthalimide part was replaced as 2-(2-amino ethoxy) ethoxyethyl group) amino group.With synthetic N-(2,2-diethoxy ethyl)-4-fourth amino-1,8 naphthalimide in add under the identical condition of butylamine, by add 2,2 '-(ethylenedioxy) two (ethamine) and carry out this final step.
Amino ethoxy F-hexa-C two-borate:
Figure A0280600700301
N-2-[5-benzyl-5-[2-(borono) benzyl] amino hexyl]-[2-(borono) benzyl] aminoethyl-4-[2-(2-amino ethoxy) ethoxyethyl group] amino-1,8-naphthalimide (aminoethoxyF-hexa-C bis-boronate)
With N-benzyl-1, the 6-diamino hexane is as the spouse of diamines coupling, with being similar to N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono) benzyl]-amino hexyl]-[2-(borono) benzyl] aminoethyl-4-[2-(2-amino ethoxy) ethoxyethyl group] amino-1, the mode of 8-naphthalimide (amino ethoxy F-hexa-Qbis-boronate) prepares this compound.
The regulation and control of fluorescence
The regulation and control of glucose have been determined to the fluorescence of three kinds of compounds preparing in the present embodiment.Fig. 2 shows amino ethoxy F-hexa-Q two-borate indicator (0.197mM) and the standardized fluorescent emission (I/Io at 535nm) of amino ethoxy F-hexa-C-hypoboric acid ester contrast indicator in comprising the 70/30MeOH/PBS solution of 0-20mM glucose.Utilize ShimadzuRF-5301 spectrofluorimeter spectra re-recorded, wherein be activated at 450nm; Excitation slit 1.5nm; Emission slit 1.5nm; Environment temperature.Error line is the standard deviation of each data point two-fold value.
Data show: the influence that the fluorescence of hexa-C indicator compound is not existed by glucose, and the fluorescence of hexa-Q indicator compound is subjected to the influence of glucose widely in the 0-10mM scope.It is believed that is not having under the situation of glucose, and pliable and tough relatively hexa-methylene connects and makes that N-4-dimethylamino benzyl quencher group can be fully near the naphthalimide fluorophor in the hexa-Q compound, thus the quencher latter's fluorescence effectively.Having under the situation of glucose, two boric acid recognition units will participate in glucose and connect, and therefore will change the molecular configuration of indicator, and fluorophor is separated fully with the quencher group, so that make fluorescent emission go quencher (dequenched).
Hexa-C compound and hexa-Q compound are equal, but lack the required dimethylamino group of effective quencher naphthalimide fluorophor.Do not having under the situation of glucose, hexa-C compound emitting fluorescence, its fluorescence is unaffected when adding glucose.
The following example 3-5 has illustrated the glucose detection method, and wherein indication mechanism comprises boric acid recognition unit and catechol ligand unit.The General Principle of this method is illustrated by following formula:
Figure A0280600700311
Wherein:
To body is fluorophor, and acceptor is fluorophor or quencher group;
To selecting for body and acceptor, so as according to molecule apart from the NE BY ENERGY TRANSFER of self-supporting body in the future to acceptor;
L 1, L 2, L 3And L 4Be to have about 3-20 chemistry that connects atom to connect base independently of one another, and including but not limited to following replacement or/and non--group (aliphatic group, aromatic group, amino, amide group, sulfo group, carbonyl, ketone, sulfamoyl or the like) that replaces;
R is the glucose recognition unit that comprises one or two phenylboric acid group;
RR is the chemical group that can form reversible ester bond with the phenylboric acid derivant of R, for example, aromatic diol (for example catechol), lactate, alpha-hydroxy acid, tartrate, malic acid, glucose, diethanolamine, poly-hydroxyl ortho position glycol (all these materials can be replacements or unsubstituted), or the like;
L 3-6And P 1-2Be dispensable group, and can have an independent existence;
As linking group L 1-4Defined such, L 5And L 6Be linking group, or the polymer chain of forming by for example acrylamide, acrylate, polyglycol or other hydrophilic polymer;
With
P 1And P 2It is hydrophilic or the polymkeric substance of hydrophobic.
When R and RR can interact in free solution, perhaps when suitably being fixed on the hydrophilic polymer, it will be fully approaching each other giving body and acceptor, so that energy can be relatively effectively from being transferred to acceptor (for example, shifting or the like by FRET, collision energy) to body.When glucose is added in the described solution, it will with being connected of RR competition and R (borate), thereby the balance of RR-R=RR+R is moved right.In the time of in being free on solution or utilize long relatively and pliable and tough coupling agent when being fixed on the polymkeric substance, R-can be moved away from each other for body and RR-acceptor portion, and reduces the energy transfer efficiency of giving between body and the acceptor, thereby fluorescent emission is increased.
Embodiment 3
At N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3; the 5-dinitro) benzoyl lysine (quencher-boronic acid, adducts) exists down; glucose is to the effect of N-in the phosphate buffered saline (PBS) (5-methoxycarbonyl group-5-[3,4-dihydroxy benzenes formamido group] amyl group)-N '-(5-fluorescein base) thiocarbamide (fluorescein-catechol adduct) fluorescent emission.
Fluorescein-catechol adduct quencher-boronic acid, adducts
N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε t-B0C-lysine methyl ester:
With 3,4-dihydroxy-benzoic acid (820 milligrams, 5.3 mMs) and N-ε-t-BOC-lysine methyl ester (1.38 grams, 5.31 mMs) are dissolved among 50 milliliters of EtOAc/THF (1/1, anhydrous).Dicyclohexylcarbodiimide (1.24 grams, 6 mMs) is added in this solution.Reaction mixture was stirred 24 hours, filter, and solvent evaporated.The solid that obtains is dissolved in (200mM, pH=6.5) 2 * 50 milliliters extract among the EtOAc (50 milliliters) and with phosphate buffer.With salt solution ethyl acetate solution is washed, separate, use Na 2SO 4Drying, and evaporation obtain 1.89 solids (90% yield) that restrain.By purify this compound and be used for next step of TLC.
Figure A0280600700341
N-α-(3,4-dihydroxy benzenes formoxyl)-lysine methyl ester trifluoroacetate:
With the CH of N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-t-BOC-lysine methyl ester (840 milligrams, 2.12 mMs) with 10 milliliters 2Cl 2, the tri isopropyl silane of 3 milliliters trifluoroacetic acid and 1 milliliter mixes.After stirred overnight at room temperature, this solution is evaporated, with ether the residue that obtains is washed, and under vacuum, carry out drying, obtain 808 milligrams (93%).
HPLC:HP 1100 high pressure liquid chromatographs, 5 * 100 millimeters NovaPak HR of water C18 post, 0.100 milliliter of injection, 0.75 ml/min, 2 milliliters of infusion circuits, detect wavelength 370nm, A=water (0.1%HFBA) and B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 10.78 minutes residence time.
N-(5-methoxycarbonyl group-5-[3,4-dihydroxy benzenes formamido group] amyl group)-N '-(5-fluorescein base) thiocarbamide:
With N-α-(3,4-dihydroxy benzenes formoxyl)-lysine methyl ester trifluoroacetate (60 milligrams, 0.146 mM), fluorescein isothiocynate (50 milligrams, 0.128 mM), and diisopropylethylamine (129 milligrams, 1 mM) mixes with 1 milliliter of dry DMF.Reaction mixture was stirred 5 hours, then solvent evaporated.Adopt silicon dioxide (10 gram) chromatographic purification, wherein use CH 2Cl 2/ MeOH (80/20 volume) is as eluent.Isolate product-68 milligram, (77% productive rate).
FAB MS: for C 35H 31N 3O 10S, calculated value are M=685; Measured value is M+1=686.
HPLC:HP 1100 high pressure liquid chromatographs, 5 * 100 millimeters NovaPak HR of water C18 post, 0.100 milliliter of injection, 0.75 ml/min, 2 milliliters of infusion circuits, detect wavelength 370nm, A=water (0.1%HFBA) and B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 16.59 minutes residence time.
N-α-(the 3-borate closes-the 5-nitro) benzoyl)-N-ε-t-BOC-lysine methyl ester:
With (536 milligrams of (3-carboxyl-5-nitrobenzophenone) boric acid; 2.54 mM), N-ε-t-BOC-lysine methyl ester hydrochloride (776 milligrams, 2.61 mMs); and diphenyl phosphoryl azide (718 milligrams, 2.6 mMs) mixes with 5 milliliters of dry DMF.Diisopropylethylamine (1.3 milliliters, 7.5 mMs) is added in the DMF solution.Then, in room temperature to this solution stirring 24 hours.Vaporising under vacuum falls DMF, residue is dissolved among 50 milliliters the EtOAc, and uses H 2O (3 * 50 milliliters) extracts EtOAc solution.After the salt solution extraction, separate organic phase, use Na 2SO 4Drying, and solvent evaporated obtain 880 milligrams product (76% yield).According to present appearance, this product is proceeded test.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mm NovaPak HR C18 post, 0.050 milliliter of injection, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 17.87 minutes residence time.
Figure A0280600700361
N-α-(the 3-borate closes-the 5-nitro) benzoyl-lysine methyl ester trifluoroacetate:
With the CH of N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-t-BOC-lysine methyl ester (800 milligrams, 1.76 mMs) with 10 milliliters 2Cl 2, the tri isopropyl silane of 3 milliliters trifluoroacetic acid and 1 milliliter mixes.After at room temperature stirring is spent the night, this solution is evaporated, with ether the residue that obtains is washed, and under vacuum, carry out drying.Obtain 715 milligrams (87%).According to present appearance, this product is proceeded test.
N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine methyl ester.
In the dark, under 25 ℃ with N-(the 3-borate closes-the 5-nitro) benzoyl-lysine methyl ester trifluoroacetate (0.198 gram, 0.42 mM); DIEA (0.167 gram; 0.225 milliliter, 1.29 mMs, 3.05 gram equivalents); 4-dimethylamino-3; 5-dinitrobenzoic acid (0.120 gram, 0.47 mM, 1.11 gram equivalents) and diphenyl phosphoryl azide (0.130 gram; 0.47 mM, 1.11 gram equivalents) solution stirring in 3 milliliters of DMF 23 hours.At this moment wait, add 50 milliliters of EtOAc, and washing in 100mM phosphate buffer (pH6.5) (2 * 20 milliliters), 1 * 25 milliliter of NaCl (saturated aqueous solution) washs this solution then.On anhydrous sodium sulfate, organic extract is carried out drying, filter and concentrate, so that obtain thick orange solids.By silica gel column chromatography (10 grammes per square metre power level gels, 0-5%CH 3OH/CH 2Cl 2) this residue is purified, thereby obtain 0.0974 the gram (39%) the yellow/orange solid.According to present appearance, this product is proceeded test.
TLC:Merck silica gel 60 plates, Rf 0.60 (80/20 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mmNovaPak HR C18 post, 0.050 milliliter of injection, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 18.91 minutes residence time.
Figure A0280600700371
N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine.
At 25 ℃, to N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine methyl ester (0.095 gram, 0.16 mM) 4 milliliters 1: 1Na 2CO 3(0.2M aqueous solution): the solution stirring among the EtOH 1 hour, stirred 1.5 hours at 45 ℃ then.At this moment wait, under vacuum, this potpourri is concentrated, add 25 milliliters of 5%TFA/CH then 2Cl 2With 2 * 10 ml waters this potpourri is washed, again with 25 milliliter 5% TFA/CH 2Cl 2Be added into organic layer.On anhydrous sodium sulfate, organic extract is carried out drying, filter and concentrate, so that obtain 0.088 gram (95%) orange powder.
FAB MS: glycerine matrix; For C 25H 29BN 6O 13(single glycerine adduct), [M] +Calculated value is 632; [M+1] +Measured value is 633.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mmNovaPakHRC18 post, 0.050 milliliter of injection, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 17.66 minutes residence time.
Fluorescence regulation and control
Fig. 3 shows the fluorescent emission (I at 518nm place) of fluorescein-catechol adduct in the PBS solution that comprises 30 μ M quencher-boronic acid, adducts of 2 μ M.The concentration of glucose changes between 0-160mM.Utilize Shimadzu RF-5301 spectrofluorimeter spectra re-recorded, wherein be activated at 495nm; Excitation slit 3nm; Emission slit 5nm; Low PMT sensitivity, environment temperature.To reduce by adding glucose quencher ability.
Embodiment 4
At N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4 dimethylaminos-3; the 5-dinitro) benzoyl lysine (quencher-boronic acid, adducts) exists down; glucose is to the effect of the fluorescent emission of the N-α in the phosphate buffered saline (PBS)-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino naphthalene-1-sulphonyl) lysine (DANSYL-catechol adduct).
Figure A0280600700381
DANSYL-catechol adduct quencher-boronic acid, adducts
Figure A0280600700382
N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino-naphthalene-1-sulphonyl)-lysine methyl ester:
N-α-(3,4-dihydroxy benzenes formoxyl)-lysine methyl ester trifluoroacetate (205 milligrams, 0.5 mM is referring to synthesizing of embodiment 3) and dansyl Cl (162 milligrams, 06 mM) are mixed with 2 milliliters of dry DMF.Diisopropylethylamine (224 milligrams, 1.7 mMs) is added in the DMF solution.At room temperature to this solution stirring 5 hours then, vaporising under vacuum falls DMF.Make residue stand silica gel chromatography (CH 2Cl 2/ MeOH, 98/2 volume).Resulting product is yellow solid-240 milligram (90% productive rate).
FAB MS: for C 29H 31N 3O 7S, calculated value are M=529; Measured value is M + 1=530.
HPLC:HP 1100 high pressure liquid chromatographs, 5 * 100 millimeters NovaPak HR of water C18 post, 0.100 milliliter of injection, 0.75 ml/min, 2 milliliters of infusion circuits, detect wavelength 370nm, A=water (0.1%HFBA) and B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 15.45 minutes residence time.
N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino-naphthalene-1-sulphonyl)-lysine:
Na with N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino-naphthalene-1-sulphonyl)-lysine methyl ester (200 milligrams, 0.38 mM) and 250 milligrams 2CO 3EtOH/H with 10 milliliters 2O (1/1 volume) mixes.At 55 ℃ potpourri was stirred 6 hours.Vaporising under vacuum falls solvent, and adds 1 milliliter trifluoroacetic acid, with the excessive alkali that neutralizes, 50 milliliters EtOAc is added in the potpourri, and uses H 2O (2 * 40 milliliters) extracts solution.Separate organic phase, carry out drying with anhydrous sodium sulfate, evaporation is so that obtain 190 milligrams of solids (productive rate 97%).
HPLC:HP 1100 high pressure liquid chromatographs, 5 * 100 millimeters NovaPak HR of water C18 post, 0.100 milliliter of injection, 0.75 ml/min, 2 milliliters of infusion circuits, detect wavelength 370nm, A=water (0.1%HFBA) and B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 14.26 minutes residence time.
N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzene first
Acyl lysine is referring to the embodiment that is used to synthesize.
Fluorescence regulation and control
Fig. 4 shows the fluorescent emission (I at the 545nm place) of red sulphonyl in the PBS that comprises 120 μ M quencher-boronic acid, adducts-catechol adduct 30 μ M solution.The concentration of glucose changes between 0-120mM.Utilize Shimadzu RF-5301 spectrofluorimeter spectra re-recorded, wherein be activated at 350nm; Excitation slit 3nm; Emission slit 5nm; High PMT sensitivity, environment temperature.To reduce by adding glucose quencher ability.
Embodiment 5
Glucose is to comprising N-α-(3; 4-dihydroxy benzenes formoxyl)-effect of the acrylamide gel fluorescent emission of N-ε-(5-dimethylamino naphthalene-1-sulphonyl)-lysine N-3-(methacrylamido) propyl group carboxylic acid amides (red sulphonyl-catechol monomer) and N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine N-3-(methacrylamido) propyl group carboxylic acid amides (quencher-boric acid monomer).
Red sulphonyl-catechol monomer quencher-boric acid monomer
N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino-naphthalene-1-sulphonyl)-lysine N-3-(methacrylamido)-propyl group carboxylic acid amides:
With N-α-(3,4-dihydroxy benzenes formoxyl)-N-((5-dimethylamino-naphthalene-1-sulphonyl)-lysine (and 75 milligrams, 0.15 mM; About synthetic, referring to embodiment 4), 3-aminopropyl methacryl amine hydrochlorate (30 milligrams, 0.17 mM), diisopropylethylamine (0.1 milliliter, 0.5 mM) and 2 milliliters of dry DMF are mixed.1-[3-(dimethylamino)-propyl group]-3-ethyl carbodiimide hydrochloride (40 milligrams, 0.2 mM) is dissolved in 2 milliliters anhydrous CH 2Cl 2In.Mix DMF and CH 2Cl 2And at room temperature stirred 20 hours.Vaporising under vacuum falls solvent, and makes residue stand silicon dioxide (7 gram) chromatography, thereby produces 18 milligrams product (19% yield).
FAB MS: for C 32H 41N 5O 7S, calculated value are M=640; Measured value is M + 1=640.
HPLC:HP 1100 high pressure liquid chromatographs, 5 * 100 millimeters NovaPak HR of water C18 post, 0.100 milliliter of injection, 0.75 ml/min, 2 milliliters of infusion circuits, detect wavelength 370nm, A=water (0.1%HFBA) and B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 14.78 minutes residence time.
N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine N-3-(methacrylamido) propyl group-carboxylic acid amides.
At 25 ℃, in the dark, with 3-aminopropyl methacryl amine hydrochlorate (0.013 gram, 0.073 mM, 1.2 gram equivalent), DIEA (0.025 gram, 0.034 milliliter, 0.19 mM, 3.2 gram equivalent), N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine (0.035 gram, 0.061 mM; For synthesizing referring to embodiment 3), diphenyl phosphoryl azide (0.019 gram, 0.015 milliliter, 0.069 mM, 1.1 gram equivalents) and the solution stirring of about 2 milligrams of BHT in 1 milliliter of dry DMF 23.5 hours.At this moment wait, add 60 milliliters of EtOAc, and wash with 200mM phosphate buffer (pH6.5) (2 * 20 milliliters), 1 * 20 milliliter of NaCl (saturated aqueous solution) washs this solution then.On anhydrous sodium sulfate, organic extract is carried out drying, filter and concentrate, so that obtain orange solids.This solid is pulverized and drying with ether, to obtain the orange powder of 0.028 gram (65%).
FAB MS: glycerine matrix; For C 32H 41BN 8O 13(single glycerine adduct), calculated value [M] +756;
Measured value [M+1] +757.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mmNovaPakHRC18 post, 0.050 milliliter of injection, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 450nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 17.98 minutes residence time.
Comprise N-α-(3; 4-dihydroxy benzenes formoxyl)-preparation of the acrylamide gel of N-ε-(5-dimethylamino naphthalene-1-sulphonyl)-lysine N-3-(methacrylamido) propyl group carboxylic acid amides and N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine N-3-(methacrylamido) propyl group carboxylic acid amides:
Preparation acrylamide (20%wt.) and N, the solution of N '-methylene diacrylamine (0.6%wt.) in ethylene glycol.With N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino naphthalene-1-sulphonyl)-lysine N-3-(methacrylamido) propyl group carboxylic acid amides (0.75 milligram, 1.6 * 10 -6Mole); N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3; the 5-dinitro) benzoyl lysine N-3-(methacrylamido) propyl group carboxylic acid amides is (3.5 milligrams; 5 * 10-6 mole) and 30 milliliters of ammonium persulfate aqueous solutions (5%wt) mix with 0.5 milliliter of glycol monomethyl liquid solution.The solution that obtains is placed the drying box that cleans with nitrogen.(30 μ L 5%wt) are added in the monomer formulation, to quicken polymerization with N,N,N.The preparation that obtains is poured in the mould that the stainless steel partition by microslide and 100 μ constitutes.After in blanket of nitrogen, keeping 8 hours, with mould place phosphate buffered saline (PBS) (PBS) (10mM PBS, pH=7.4) in, separate microslide, and hydrogel taken out.With 100 milliliters of PBS that comprise 1mM lauryl sulfate sodium salt and 1mM EDTA sodium salt, to this hydrogel washing 3 days, solution all changed every day, use DMF/PBS (10/90 volume then, 3 * 100 milliliters) wash, use PBS (pH=7.4,3 * 100 milliliters) to wash at last.With the aquogel polymer that obtains be stored in the PBS that comprises 0.2%wt sodium azide and 1mM EDTA sodium salt (10mMPBS, pH=7.4) in.
Fluorescence regulation and control
Fig. 5 shows the fluorescent emission (I at 532nm place) of acrylamide gel (20%) in PBS that comprises the red sulphonyl of 2mM-catechol monomer and 10mM quencher-boric acid monomer.Gel (100 μ m thickness) is applied to PMMA than in the colour circle.The concentration of glucose changes between 0-200mM.Utilize Shimadzu RF-5301 spectrofluorimeter spectra re-recorded, wherein be activated at 350nm; Excitation slit 3nm; Emission slit 10nm; High PMT sensitivity, 37 ℃.To reduce by adding glucose quencher ability.
Embodiment 6
Figure A0280600700441
Hypofluorescence utmost point hypofluorescence hyperfluorescence
Glucose is for 3, and there is the preparation of the soluble anthracene hypoboric acid of the effect PBS derivant of the fluorescence of anthracene two-boronic acid derivatives down in the 4-dihydroxy-benzoic acid:
9,10-two [[2-(uncle-butoxy carbonyl) ethylamino-] methyl]-anthracene
In the dark, at 23 ℃, with Beta-alanine tert-butyl ester hydrochloride (3.06 grams, 16.8 mMs, 5.09 gram equivalent), DIEA (4.27 grams, 5.75 milliliters, 33.0 mMs, 10.00 gram equivalent) and 9,10-two (chloromethyl) anthracene (0.910 gram, 3.31 mMs) is at 75 milliliters of CHCl 3In solution stirring 93 hours.At this moment wait, this solution is filtered, and with the NaHCO of 1 * 40 milliliter and 2 * 60 milliliters 3(saturated aqueous solution) washs.On anhydrous sodium sulfate, organic extract is carried out drying, filter and concentrate, so that obtain thick yellow solid.By silica gel column chromatography (30 grammes per square metre power level gels, 0-3%CH 3OH/CH 2Cl 2) this residue is purified, thereby obtain 1.06 the gram (65%) viscosity Huang-orange material.According to present appearance, this product is proceeded test.
TLC:Merck silica gel 60 plates, Rf 0.33 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
9,10-two [N-[2-(5,5-dimethyl borine-2-yl) benzyl]-N-[2-(uncle-butoxy carbonyl) ethylamino-] methyl] anthracene
In the dark, in 23 ℃, with 9,10-two [[2-(uncle-butoxy carbonyl)-ethylamino-] methyl] anthracene (1.60 grams, 3.25 mMs), DIEA (4.45 grams, 6.00 milliliter, 34.4 mMs, 10.6 gram equivalents) and (2-2-bromomethylphenyl) boric acid neopentyl ester (4.80 grams, 17.0 mM, 5.22 gram equivalents) at 30 milliliters of CHCl 3In solution stirring 4.5 hours.At this moment wait, 45 milliliters of CHCl 3Be added in the potpourri, with 2 * 25 milliliters NaHCO 3(saturated aqueous solution) washs this potpourri.On anhydrous sodium sulfate, organic extract is carried out drying, filter and concentrate, so that obtain thick reddish oil.By alumina column chromatography (neutral alumina that 100 grams activate, 0-3%CH 3OH/CH 2Cl 2) this residue is purified, thereby obtain about 3.5 orange solids that restrain.Make this product dissolving, form one then and precipitate (DIEA-HBr salt) in vain.This solution is filtered, and concentrated filtrate obtains the orange solids of 2.72 grams (93%).According to present appearance, product (＞80% purity is passed through RP-HPLC) is proceeded test.
The alkaline oxygenated aluminium sheet of TLC:Merck, Rf 0.66 (95/5 CH 2Cl 2/ CH 3OH), utilize UV (254/366) to observe.
HPLC condition: HP 1100HPLC chromatograph, Vydac 201TP 10 * 250mm post, 0.100 milliliter injects, 2 ml/min detect wavelength 370nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA) gradient 10%B2 minute, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 23.9 minutes residence time.
Figure A0280600700461
9,10-two [N-(2-borono benzyl)-N-[3-(propiono) amino]-methyl] anthracene
In the dark, at 23 ℃,, 10-two [N-[2-(5,5-dimethyl borine-2-yl) benzyl]-N-[2-(uncle-butoxy carbonyl) ethylamino-with 9]-methyl] anthracene (0.556 gram, 0.620 mM) is at 5 milliliters of 20%TFA/CH 2Cl 2In solution stirring 25 hours.At this moment, flow down at nitrogen reaction mixture is concentrated.With 3 * 10 milliliters ether this residue is pulverized.Make remaining solid drying in a vacuum, to obtain the fluffy yellow powder of 0.351 gram (87%).
FAB MS: glycerine matrix; For C 42H 46B 2N 2O 10(two glycerine adducts), calculated value [M] +760; Measured value [M+1] +760.
HPLC:HP 1100HPLC chromatograph, water 5 * 100mm NovaPak HR C18 post, 0.025 milliliter of injection, 0.75 ml/min, 1.5 the milliliter infusion circuit detects wavelength 360nm, A=water (0.1%HFBA), B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B18 minute, 80-100%B2 minute, 100%B2 minute, 16.7 minutes residence time.
Fluorescence regulation and control
Fig. 6 has shown 3, and the 4-dihydroxy-benzoic acid is to the effect of anthracene hypoboric acid derivant (the 40 μ M) fluorescence intensity (450nm) among the PBS of preparation in this embodiment.Utilize ShimadzuRF-5301 spectrofluorimeter spectra re-recorded, wherein be activated at 370nn; Excitation slit 3nm; Emission slit 3nm; High PMT sensitivity, environment temperature.Anthracene two-boronic acid derivatives is launched low-level fluorescence, and it will be owing to 3, the existence of 4-dihydroxy-benzoic acid and quencher effectively.
Fig. 7 has shown that 3 4-dihydroxy-benzoic acid (200 μ M) exists down, and the standardized fluorescence intensity (430nm) of the anthracene hypoboric acid derivant of present embodiment (40 μ M) is with the variation (Diamond spot) of concentration of glucose among the PBS; And the standardized fluorescence intensity (430nm) of identical indicator (40 μ M) is with the variation (square dot) of concentration of glucose among the PBS.The concentration of glucose changes between 0-25mM.Utilize Shimadzu RF-5301 spectrofluorimeter spectra re-recorded, wherein be activated at 370nm; Excitation slit 3nm; Emission slit 5nm; Low PMT sensitivity, environment temperature.Do not having 3, under the situation of 4-dihydroxy-benzoic acid quencher, glucose is being added in the anthracene hypoboric acid derivant, fluorescence will be strengthened.Having 3, under the situation of 4-dihydroxy-benzoic acid quencher, glucose is being added in the anthracene hypoboric acid derivant, fluorescence will significantly be strengthened.It is believed that this glucose will replace 3 from the boric acid recognition unit, 4-dihydroxy-benzoic acid quencher, the result increases fluorescence.In this embodiment, 3,4-dihydroxy-benzoic acid base not only plays detection system quencher part, but also plays a part and the interactional ligand of recognition unit unit.
Embodiment 7
Figure A0280600700471
A.1,4-two [[the amino fourth amino of 4-(uncle-butoxy carbonyl)] methyl] benzene:
With terephthalaldehyde (0.253 gram, 1.89 mMs), uncle N--Boc-butanediamine (0.71 gram, 3.77 mMs) and sodium sulphate (5.5 restrain 40 mMs) mix with 25 milliliters absolute methanol.At room temperature, this potpourri was stirred 24 hours, filter out sodium sulphate, and add NaBH 4(1.5 grams, 40 mMs).After 4 hours, with 100 milliliters of ether diluted mixture things and filter.Make residue that solvent evaporation obtains after falling through silica gel chromatography, wherein utilize CH 2Cl 2/ MeOH/Et 3N (85/15/5 volume %) is as eluent.Institute's separated products is white solid (0.77 gram, 86% yield).In next step, utilize this material according to present appearance.
Figure A0280600700481
B.1,4-two [N-[2-(pinacol) borono benzyl]-amino fourth amino of N-[[4-(uncle-butoxy carbonyl)] methyl] benzene:
With 2-2-bromomethylphenyl boric acid, pinacol ester (1.4 grams, 4.7 mMs), 1,4-two [[the amino fourth amino of 4-(uncle-butoxy carbonyl)] methyl] benzene (0.74 gram, 1.56 mM), and N, N-diisopropyl-N-ethamine (1.8 milliliters, 10 mMs) is dissolved in 20 milliliters CH 2Cl 2In.At room temperature to this solution stirring 24 hours, solvent evaporated, and with hexane/ether (50/50 volume, 3 * 10 milliliters) residue is washed.(silicon dioxide, 90/10 volume CH2Cl2/MeOH) is purified to product by column chromatography again.Get product 1.18 grams (yield 83%).
Figure A0280600700491
C.1,4-two [the amino fourth amino of N-(2-borono benzyl)-N-[4-] methyl] benzene two (trifluoroacetic acid) salt:
With 1,4-two [N-[2-(pinacol) borono the benzyl]-amino fourth amino of N-[[4-(uncle-butoxy carbonyl)] methyl] benzene (1.1 grams, 1.2 mMs) is dissolved in 20 milliliters CH of the tri isopropyl silane of the TFA that comprises 20% volume and 5% volume 2Cl 2In the solution.To solution stirring 12 hours, and solvent evaporated, under vacuum in 50 ℃ to dry 24 hours of residue.Quantitative yield.
FAB MS: for C 42H 64B 2N 4O 4, calculated value M +=710 (two pinacol esters), measured value M + 2=712.
HPLC:HP 1100 high pressure liquid chromatographs, 5 * 100 millimeters NovaPak HR of water C18 post, 0.100 milliliter of injection, 0.75 ml/min, 2 milliliters of infusion circuits, detect wavelength 280nm, A=water (0.1%HFBA) and B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 14.6 minutes residence time.
D.3,4-dihydroxy-9,10-dioxo-2-anthracene sulfonic acid chloride:
With 3,4-dihydroxy-9, (1.4 grams 3.9mM) mix with 30 milliliters chlorosulfonic acid 10-dioxo-2-rylnthracene sulfonin sodium salt, and 90 ℃ of heating 5 hours, then, make this solution be cooled to 0 ℃, and pour 100 into and restrain and ice.Using CH 2Cl 2After the solution extraction that (3 * 100 milliliters) dissolve ice, mix the dichloromethane extraction agent, use Na 2SO 4Carry out drying and evaporation, to produce the solid (yield 66%) of 0.87 gram.
E.1-[N-the amino fourth amino of (2-borono benzyl)-N-[4-] methyl]-4-[N-(2-borono benzyl)-N-[4-[(3,4-dihydroxy-9,10-dioxo-2-anthracene) sulfonamido] fourth amino] methyl]-the benzenesulfonamide trifluoroacetate acetate:
With 3,4-dihydroxy-9,10-dioxo-2-anthracene sulfonic acid chloride (0.095 gram, 0.28 mM) is dissolved in 3 milliliters anhydrous CH 3Among the CN, and drop to 1,4-two [the amino fourth amino of N-(2-borono benzyl)-N-[4-] methyl] benzene two (trifluoroacetate) (1.06 grams, 1.37 mMs) and N, N-diisopropyl-N-ethamine (1 milliliter, 5.8 mMs) is at 5 milliliters of anhydrous CH 3In the solution among the CN.After stirring 4 hours, solvent evaporated is also carried out drying to residue under vacuum.Residue is dissolved in 10 milliliters CH 3Solvent evaporated among the CN/TFA (80/20 volume %) and once more.Water (10 milliliters) is added in this residue, and flask is carried out 20 minutes sonicated, then filter the brown solid that comprises product.Further use the HPLC of preparation to purify: HP 1100 high pressure liquid chromatographs, 25 * 100 millimeters NovaPak HR of water C18 post, 1.00 milliliters of injections, 5 ml/min flow velocitys, 2 milliliters of infusion circuits, detect wavelength 470nm, A=water (0.1%HFBA) and B=MeCN (0.1%HFBA), gradient 10%B2 minute, 10-80%B in 18 minutes, 80-100%B in 2 minutes, 100%B2 minute, 18.5 minutes residence time.Obtain 198 milligrams of products (yield 79%).Just in MeOH/PBS (1/1, volume ratio) solution (pH=7.4), the interaction of this compound and D-glucose is tested, interaction is assessed by monitoring to absorption spectrum.
F.1-[N-(2-borono benzyl)-N-[4-(methacrylamido) fourth amino] methyl]-4-[N-(2-borono benzyl)-N-[4-[(3,4-dihydroxy-9,10-dioxo-2-anthracene) sulfonamido] fourth amino] methyl]-benzene:
With the amino fourth amino of 1-[N-(2-borono benzyl)-N-[4-] methyl]-4-[N-(2-borono benzyl)-N-[4-[(3,4-dihydroxy-9,10-dioxo-2-anthracene) sulfonamido] fourth amino] methyl] the benzenesulfonamide trifluoroacetate acetate (30 milligrams, 3.34 * 10 -5Mole) is dissolved in 1 milliliter the absolute methanol.Interpolation is according to the methacrylic acid NHS ester of J.Am.Chem.Soc. (1999,121 (15), 3617) preparation
Figure A0280600700511
(10 milligrams, 5.46 * 10 -5Mole), add 0.01 milliliter Et then 3N.With this solution stirring 10 hours.Vaporising under vacuum falls solvent, and water washs solid.RP-HPLC the analysis showed that: do not have starting material in solid.Under vacuum, the solid that obtains is carried out drying, and aggregate into hydrogel thin film according to present appearance.
G. comprise 1-[N-(2-borono benzyl)-N-[4-(methacrylamido) fourth amino] methyl]-4-[N-(2-borono benzyl)-N-[4-[(3,4-dihydroxy-9,10-dioxo-2-anthracene) sulfonamido] fourth amino] methyl]-preparation of the N-N-DMAA hydrogel thin film of benzene:
Preparation N,N-DMAA (40% weight) and N, N '-methylene diacrylamine (0.8% weight) and the solution of D-fructose (200mM) in DMF.With 1-[N-(2-borono benzyl)-N-[4-(methacrylamido) fourth amino] methyl]-4-[N-(2-borono benzyl)-N-[4-[(3,4-dihydroxy-9,10-dioxo-2-anthracene) sulfonamido] fourth amino] methyl]-benzene (30 milligrams) is dissolved in the 0.5 milliliter of DMF solution that comprises monomer and D-fructose.Moisture ammonium persulfate (20 μ L, 5% weight) is mixed with this prescription.The solution that obtains is placed the glove box that cleans with nitrogen.(20 μ L 5%wt) are added in the monomer prescription, to quicken polymerization with the aqueous solution of N,N,N.The preparation that obtains is poured in the mould that the stainless steel partition by microslide and 100 μ M constitutes.After in blanket of nitrogen, keeping 8 hours, with mould place phosphate buffered saline (PBS) (10mM pi, pH=7.4) in, separate microslide, and hydrogel taken out.With 100 milliliters of the phosphate buffered saline (PBS)s (PBS) that comprises 1mM lauryl sulfate sodium salt and 1mM EDTA sodium salt, to this hydrogel washing 3 days, solution all changes every day, use DMF/PBS (10/90 volume then, 3 * 100 milliliters) wash, use PBS (pH=7.4,3 * 100 milliliters) to wash at last.With the aquogel polymer that obtains be stored in the PBS that contains 0.2%wt sodium azide and 1mM EDTA sodium salt (10mM PBS, pH=7.4) in.
H.D-glucose is to comprising 1-[N-(2-borono benzyl)-N-[4-(methacrylamido) fourth amino] methyl]-4-[N-(2-borono benzyl)-N-[4-[(3,4-dihydroxy-9,10-dioxo-2-anthracene) sulfonamido] fourth amino] methyl]-fluorescence of the N-N-DMAA gel of benzene and the effect of absorptance:
In being equipped with the Shimadzu RF-5301 PC spectrofluorimeter of variable temperature annex, carry out this experiment.The N,N-DMAA hydrogel is invested one to be glued on the glass sheet in the PMMA fluorescence pond with miter angle.Fill this pond with the PBS that comprises various concentration D-glucose (pH=7.4) solution.Before absorptance and fluorescence intensity are measured, with this pond 37 ℃ of balances 30 minutes.The excitation wavelength of measuring fluorescence intensity is arranged on 470nm, and slit-widths is 3/3nm, highly sensitive PMT.Utilize HP 8453 instruments that the absorption spectrum of hydrogel thin film is measured, in each the measurement, carry out blank correction with the absorptance at 690nm place.
The results are shown among Fig. 8-10.Fig. 8 shows the absorption spectrum of indicator in containing the PBS/ methyl alcohol of different concentration of glucose.Fig. 9 shows the absorptivity of the indicator gel (A (565nm)/A (430nm)) that contains different concentration of glucose.Figure 10 shows the standardization fluorescence (I/Io) of variable concentrations glucose at the 550nm place.

Claims (24)

1. the existence of polyhydroxy analysans or the method for concentration in the test samples, this method comprises:
A) sample is exposed to indication mechanism, described system has:
I) can be with first recognition unit of reversible manner and described analysans formation covalent bond, with can form second recognition unit (A) of covalent bond with reversible manner and the described analysans that is bonded on first recognition unit, or can be when not having described analysans with reversible manner and the interactional ligand of first recognition unit unit (B); Described ligand unit comprises the mark that generation can detect characteristic also not essentially, the described characteristic that detects is regulated and control by the interaction of ligand unit and recognition unit, the indication mechanism part that wherein comprises described first recognition unit covalently or non--covalently be connected on the part that comprises described second recognition unit or described ligand unit in the indication mechanism; With
Ii) comprise following detection system one of at least: generation can detect the donor/acceptor system (A) of characteristic, when described indication mechanism is exposed in the described analysans, this system changes in the mode of concentration-dependence, or the described ligand unit (B) that is labeled; With
B) measure the described any change that detects characteristic, be determined at the existence or the concentration of analysans described in the described sample thus.
2. the process of claim 1 wherein that indication mechanism has two recognition units at analysans at least.
3. the method for claim 2, wherein analysans is a sugar, and each recognition unit is independently selected from boric acid, borate ion, arsenious acid, arsenous anion ion, telluric acid, tellurate radical ion, germanic acid, germanic acid radical ion and combination thereof.
4. the method for claim 3, wherein analysans is a glucose, and each recognition unit comprises one or more boronate.
5. the process of claim 1 wherein that indication mechanism has recognition unit and the ligand unit at analysans.
6. the method for claim 5, wherein analysans is a sugar, and recognition unit comprises one or more following materials: boric acid, borate ion, arsenious acid, arsenous anion ion, telluric acid, tellurate radical ion, germanic acid or germanic acid radical ion.
7. the method for claim 6, wherein analysans is a glucose, and recognition unit comprises one or more boronate.
8. the method for claim 5, wherein the ligand unit is the part that can form ester bond with recognition unit.
9. the method for claim 8, the ligand unit compound that is selected from aromatic diol, lactate, alpha-hydroxy acid, tartrate, malic acid, diethanolamine, beta-alkamine, glucose and polyol and contains vicinal hydroxyl groups wherein, described these materials can be to replace or unsubstituted.
10. the process of claim 1 wherein that detection system comprises to body/receptor system.
11. the method for claim 10, wherein detection system comprises fluorophor and quencher group, and wherein when described indication mechanism was bonded on the described analysans, described fluorophor was by quencher or gone quencher.
12. the process of claim 1 wherein that detection system comprises the described ligand unit that is labeled.
13. the method for claim 12, the wherein said ligand unit that is labeled comprises fluorophor, and the fluorescence of described fluorophor combines by described indication mechanism and described analysans.
14. the method for claim 10, wherein detection system comprises at least two different fluorophors, and the fluorescence of wherein said fluorophor is regulated and control by the interaction of described indication mechanism and described analysans.
15. the process of claim 1 wherein that sample is a physiological fluid.
16. the method for claim 15, wherein physiological fluid is selected from liquid, lymph, tears, sweat and the physiologic buffer of blood, blood plasma, serum, interstitial fluid, celiolymph, urine, saliva, intraocular.
17. the process of claim 1 wherein that indication mechanism is exposed to the sample in the solution.
18. the process of claim 1 wherein that indication mechanism is fixed on the solid phase carrier or is fixed on its inside.
19. the method for claim 18, wherein solid phase carrier is a polymeric matrix.
20. the process of claim 1 wherein that indication mechanism combines with implantable device, and wherein step (a) is carried out in vivo.
21. the process of claim 1 wherein that measuring process is carrying out under the environment temperature basically.
22. the method for claim 21, wherein temperature is high to about 80 ℃.
23. the process of claim 1 wherein that indication mechanism comprises the residue that is selected from following compound:
N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono)-benzyl] amino hexyl]-[2-(borono) benzyl] aminoethyl-4-fourth amino-1, the 8-naphthalimide;
N-2-[4-(N-4-dimethylamino benzyl)-[2-(borono)-benzyl] aminomethyl] benzyl-[2-(borono) benzyl] aminoethyl-4-fourth amino-1, the 8-naphthalimide;
N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono)-benzyl] amino hexyl]-[2-(borono) benzyl] aminoethyl-4-[2-(2-amino ethoxy) ethoxyethyl group) amino-1, the 8-naphthalimide;
N-(5-methoxycarbonyl group-5-[3,4-dihydroxy benzenes formamido group] amyl group)-N '-(5-fluorescein base) thiocarbamide;
N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine;
N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino-naphthalene-1-sulphonyl)-lysine;
N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino-naphthalene-1-sulphonyl)-lysine
N-3-(methacrylamido)-propyl group formamide; With
N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine N-3-(methacrylamido) propyl group-carboxylic acid amides.
24. indication mechanism that comprises the residue that is selected from following compound:
N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono)-benzyl] amino hexyl]-[2-(borono) benzyl] aminoethyl-4-fourth amino-1, the 8-naphthalimide;
N-2-[4-(N-4-dimethylamino benzyl)-[2-(borono)-benzyl] aminomethyl] benzyl-[2-(borono) benzyl] aminoethyl-4-fourth amino-1, the 8-naphthalimide;
N-2-[5-(N-4-dimethylamino benzyl)-5-[2-(borono)-benzyl] amino hexyl]-[2-(borono) benzyl] aminoethyl-4-[2-(2-amino ethoxy) ethoxyethyl group) amino-1, the 8-naphthalimide;
N-(5-methoxycarbonyl group-5-[3,4-dihydroxy benzenes formamido group] amyl group)-N '-(5-fluorescein base) thiocarbamide:
N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine;
N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino-naphthalene-1-sulphonyl)-lysine;
N-α-(3,4-dihydroxy benzenes formoxyl)-N-ε-(5-dimethylamino-naphthalene-1-sulphonyl)-lysine N-3-(methacrylamido)-propyl group formamide; With
N-α-(the 3-borate closes-the 5-nitro) benzoyl-N-ε-(4-dimethylamino-3,5-dinitro) benzoyl lysine N-3-(methacrylamido) propyl group-carboxylic acid amides.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN112601491A (en) * 2018-06-27 2021-04-02 普罗菲尤萨股份有限公司 Near-infrared glucose sensor
US11866588B2 (en) 2016-12-21 2024-01-09 Profusa, Inc. Polymerizable near-IR dyes

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2235738C (en) 1995-11-22 2005-07-26 Minimed, Inc. Detection of biological molecules using chemical amplification and optical sensors
WO1998022820A1 (en) 1996-11-21 1998-05-28 Lawrence Livermore National Laboratory Detection of biological molecules using boronate-based chemical amplification and optical sensors
US6673625B2 (en) 1999-09-15 2004-01-06 The Regents Of The University Of California Saccharide sensing molecules having enhanced fluorescent properties
US6682938B1 (en) 1999-09-15 2004-01-27 The Regents Of The University Of California Glucose sensing molecules having selected fluorescent properties
US6387672B1 (en) 2000-12-04 2002-05-14 Beckman Coulter, Inc. Photo-induced electron transfer fluorescent sensor molecules
US6627177B2 (en) * 2000-12-05 2003-09-30 The Regents Of The University Of California Polyhydroxyl-substituted organic molecule sensing optical in vivo method utilizing a boronic acid adduct and the device thereof
US7905134B2 (en) 2002-08-06 2011-03-15 The Regents Of The University Of California Biomarker normalization
AU2003233449B2 (en) * 2002-08-06 2009-07-02 The Regents Of The University Of California Tear film osmometry
US7810380B2 (en) 2003-03-25 2010-10-12 Tearlab Research, Inc. Systems and methods for collecting tear film and measuring tear film osmolarity
WO2004096817A1 (en) * 2003-03-28 2004-11-11 Terumo Corporation Of Japan Solid-phase saccharide sensing compounds
US7297548B2 (en) * 2003-03-28 2007-11-20 Terumo Kabushiki Kaisha Solid-phase saccharide sensing compounds
US7358094B2 (en) * 2003-05-01 2008-04-15 Bell Michael L Sensor system for saccharides
JP5547061B2 (en) * 2007-06-08 2014-07-09 ザ・チャールズ・スターク・ドレイパ・ラボラトリー・インコーポレイテッド Sensors for the detection of diols and carbohydrates using boronic acid chelators for glucose
US8420665B2 (en) 2008-01-11 2013-04-16 Northwestern University Anti-cancer compounds
JP5775004B2 (en) 2009-03-03 2015-09-09 アクセス メディカル システムズ，リミティド Detection system and method for high sensitivity fluorescence analysis
EP2536335A1 (en) * 2010-02-19 2012-12-26 Glysure Ltd Intravascular glucose sensor
US20130344619A1 (en) * 2012-06-21 2013-12-26 Lightship Medical Limited Glucose sensor
KR101665046B1 (en) 2013-01-31 2016-10-11 인하대학교 산학협력단 Ratiometric fluorescent chemosensor including boronic acid binding to mercury ion selectively, preparation method thereof and detection method of mercury ion using the same
WO2014149198A1 (en) * 2013-03-15 2014-09-25 Alumend, Llc Compositions and methods of using the compositions for plaque softening
ES2828278T3 (en) 2014-09-23 2021-05-25 Tearlab Res Inc Microfluidic tear collection integration system and lateral flow analysis of analytes of interest
WO2018017842A1 (en) * 2016-07-20 2018-01-25 University Of Maryland, Baltimore Silicone hydrogel based fluorescent assay and contact lens
WO2020247662A1 (en) * 2019-06-04 2020-12-10 The Regents Of The University Of California Diboronic acid compounds and methods of making and using thereof
KR102580296B1 (en) * 2021-08-04 2023-09-19 아주대학교산학협력단 Indolizine derivative compound, composition for detecting glucose comprising the same, sensor for detecting glucose and method for detecting glucose

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4329461A (en) * 1980-01-11 1982-05-11 Syva Company Fluorescent thyroid hormone conjugates and their uses
US5512246A (en) * 1989-09-21 1996-04-30 Anthony P. Russell Method and means for detecting polyhydroxyl compounds
US6040194A (en) * 1989-12-14 2000-03-21 Sensor Technologies, Inc. Methods and device for detecting and quantifying substances in body fluids
GB2284809B (en) * 1993-11-07 1998-04-29 Japan Res Dev Corp A fluorescent phenylboronic acid suitable for use in the detection of saccharides
US5517313A (en) * 1995-02-21 1996-05-14 Colvin, Jr.; Arthur E. Fluorescent optical sensor
JP2799837B2 (en) * 1995-03-03 1998-09-21 科学技術振興事業団 Boronic acid compounds having a binaphthyl group
CA2235738C (en) * 1995-11-22 2005-07-26 Minimed, Inc. Detection of biological molecules using chemical amplification and optical sensors
US6002954A (en) * 1995-11-22 1999-12-14 The Regents Of The University Of California Detection of biological molecules using boronate-based chemical amplification and optical sensors
US5833603A (en) * 1996-03-13 1998-11-10 Lipomatrix, Inc. Implantable biosensing transponder
US5894351A (en) * 1997-05-13 1999-04-13 Colvin, Jr.; Arthur E. Fluorescence sensing device
US5917605A (en) * 1997-05-13 1999-06-29 Colvin, Jr.; Arthur E. Fluorescence sensing device
US5910661A (en) * 1997-05-13 1999-06-08 Colvin, Jr.; Arthur E. Flourescence sensing device
EP1062516A1 (en) * 1998-03-11 2000-12-27 Sensors for Medicine and Science, Inc. Detection of analytes by fluorescent lanthanide chelates
US6387672B1 (en) * 2000-12-04 2002-05-14 Beckman Coulter, Inc. Photo-induced electron transfer fluorescent sensor molecules

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11866588B2 (en) 2016-12-21 2024-01-09 Profusa, Inc. Polymerizable near-IR dyes
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CN112601491A (en) * 2018-06-27 2021-04-02 普罗菲尤萨股份有限公司 Near-infrared glucose sensor

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